Stable heaparin solution



ological solutions.

3,030,272 Patented Apr. 17, 1962 fitice Illinois No Drawing. Filed May 26, 1960, Ser. No. 31,840 8 Claims. (Cl. 167-74) The present invention is concerned with a stable heparin solution; more particularly it is concerned with stabilizing a solutionof sodium heparin for storage in certain types of plastic containers.

Heparin is a material isolated from animal tissue and has found high acceptance as a blood anti-coagulant. As such it is used alone or it may be added to various physiologically useful solutions intended for entering the blood vessels.\ When used by itself heparin is ordinarily applied as its sodium salt which forms a colorless, clear solution. If such a solution is stored under exclusion of other chemicals, the solution remains intact almost indefinitely. However, when certain other chemicals have access to the heparin solution, e.g. from other materials included in this physiological solution or from the container material, it loses some or all of its activity within relatively short periods of time. Containers other than glass often provide such deteriorating materials, be it by surface decomposition or by a leaching effect. This decomposition is particularly evident when heparin sodium is combined with dextrose or similar physiologically often used compounds or when heparin sodium is stored in one of the recently developed unbreakable, transparent plastic containers made from resins such as polyvinylchloride.

' The decomposition of a heparin solution is particularly dangerous 'because'the decomposition products are also colorless and soluble and no visible change is observed in the heparin solution. In spite of the fact that no visible change occurs, the activity of the heparin solution initially stored in contact with foreign materials decreases at a relatively rapid pace, and after only a few months, the

original activity reduces by more than about20% even if the heparin solution has been carefully stored at room temperature or below.

It is therefore an object of the present invention to proordinary medical containers used for storage of physi- It is another object of the present invention to provide a heparin solution stable to the influences of light, temperature, and the container material. A particular object is the provision of a heparin solution retaining its activity for several years when stored in transparent plastic containers made from polyvinylchloride. Another object is the provision of a heparin solution which "remains stable in spite of the presence of other physiologically desirable ingredients. Other objects will be established as the minimum amount to give the effective 'protection to a heparin sodium solution. The upper limit 'of 0.5% also is a practical limit since higher percentages will not produce any better results and larger amounts may be objectionable to some physicians for fear of introducing excessive amounts of citric acid or sodium citrate into the patients body in combination with the indicated amount of heparin. However, citric acid and sodium citrate are both compounds easily tolerated by the human body in amounts much larger than the amounts referred to above.

The combination of sodium citrate and citric acid acts as a buffer to the heparin solution and both ingredients are easily water soluble and form colorless solutions therein.

A practical, physiological heparin solution contains about 2000 units of heparin in the form of sodium heparin per 30 cc. of solution. Such a solution, free of other ingredients, is storable indefinitely in medicinally accepted glass containers, but when stored together with dissociating ingredients or in the more recently developed transparent plastic bags, it loses a considerable amount of its activity in a matter of three months. By admixing 0.01- 0.5% of sodium citrate/ citric acid with such a sodium heparin solution, the sodium citrate/ citric acid acts as a buffer and prevents decomposition of the heparin by the influence of certain otherchemicals combined with it or from decomposition products from the plastic container.

No other effect of the sodium citrate/citric acid is observed by adding this amount of the buffer, i.e. neither the container nor the solution nor ultimately the consumers body are affected by such an amount.

For better understanding of the present invention the following examples are added. These examples are meant to be illustrations only and are not intended to limit the present invention to these specific embodiments. In all these examples, sodium heparin solutions are used which contain 40-67 U.S.P. units of heparin per milliliter of solution. The solutions are filtered prior to filling them into the containers and are subsequently heat-sterilized in the containers at 112 C. for half an hour in an autoclave. The activity of the heparin solution is measured by bioassays. Assay differences of about 5% lie within the experimental error limit. All percentages are to be understood as percent of remaining activity, the initial solution activity being arbitrarily set at 100.

EXAMPLE 1 An isotonic sodium heparin solution containing 2000 U.S.P. heparin units and 0.45 g. of sodium chloride in 50 cc. of solution is stored in medicinally accepted plasticized polyvinylchloride bags, the bags are stored at 5, 25, and 40 C. for several months and the activities measured after the following time periods.

Table 1 Period 5 C. 25 C. 40 0.

Percent Percent Percent 3 months... 87 74 6 months 86 80 12 months.-. 90 77 EMMPLE 2 The isotonic sodium heparin solution described in Example 1 is stored in glass bottles containing shredded plasticized polyvinylchloride in an amount producing the same surface area as if the heparin solution were stored in polyvinylchloride bags. A control test is run wherein the glass bottle does not contain any polyvinylchloride.

Table II Test Period Control, Sample,

percent percent 2 Weeks at 60 0..-- V 101 92 1 month at 60 C. 103 70 2 months at 60 C 65 3 months at 60 0.- 100 36 From Tables I and II it will be apparent that storing sodium heparin in glass containers does not afiect its activity, even at elevated temperatures. However, when the heparin solution is stored in contact with plasticized polyvinylchloride, the activity decreases quite rapidly.

EXAMPLE 3 (a) The-.isotonicsodiumheparin solution described in Example 1.is buttered with 0.026% sodium citrate-and 0.0006% citricacid, resulting in a pH of 6.

(b) A. similar solution is bufiiered with 0.028 1 sodiumcitrate and 0.005% citric acid,.resultingin apH of 5.4.

Bothsolutions arestored in amounts of 50 cc. in 500 cc. plasticized polyvinylchloride containers which, in turn, are placed in glass bottles. The bottles are not moisture-tight, permitting portions of the solvent to escape in form of vapors which results in. an increased heparinconcentration and higher activity.

Table III Period Control (a) (b) Percent Percent 105 107 112 139 Percent Initially 2 weeks at 60 0.- Lmonth at 60 C 2 monthsat 60 C.-.

Considering the loss of moisture during this experiment indicates that the solution as buttered in (a) remains about unchanged, whereas the buffered solution (b) retains about 83% activity, while the control sample activity reduces to 59%.

EXAMPLE 4 The above isotonic sodium heparin solution is stored in amounts of 50 cc. in 500 cc. plastic containers made from plasticized polyvinylchloride. (a) Unbuffered control. (b) Butler: 0.16% sodium citrate, 0.002% citric acid, pH 6.5.

H(c6) Buffer: 0.32% sodium citrate, 0.004% citric acid, p

H(c)5Buifer: 0.64% sodium citrate, 0.008% citric acid, p

The initial heparin activity is 104% and the containers .are stored at various temperatures as shown in the table.

Table I V Period (a) (b) (c) v 2 weeks at 60 C 05) 105 1 l3 3 months at 40 C 3 EMMPLE 5 The solution described in Example 4(b) is stored in a volume of 50 cc. in a plasticized polyvinylchloride container of 500 cc. capacity. The initial activity is 105% and the solution is stored at 25, 40 and 60 C. respectively. The following activity results are determined.

Solutions containing 2000 U.S.P. units of heparin and 2.50 g. of dextrose in 50 cc. of solution are stored under various conditions and with various bufiers in 500 cc. plasticized polyvinylchloride containers. The initial pH is 6.5 for all samples and the activity is 107% initially. The following activities are determined by bioassays.

Table V] Period Control (a) (b) (0) Percent Percent Percent Percent Sodium citrate, 0. 16 0. 24 0.32 Citric acid 0. 002 0.003 0. 004 1 month at 40 94 6 112 108 2 months at 60 19 78 92 3 months at 40 82 103 107 3 months at 60 8. 0 82 78 85 By leaving out the addition of citric acid from the above solutions (a), (b) and (c), substantially the same results are obtained with the only exception that thepH is slightly higher.

EXAMPLE 7 Sodium heparin solutions containing 2000 U.S.P. units of heparin, 0.1125 g. of sodium chloride and 2.5 g. of dextrose per 50 cc. of solution are stored in 150 cc. glass containers containing shredded plasticized polyvinylchloride with a surface area similar to a plastic container of the same volume. Tests (a) and (b) contain the solutions of Examples 6(b) and 6(a) respectively and test sample .(c) contains 0.48% sodium citrate and 0.006% citric acid.

Table VII Period Control (a) (b) r (0) Percent Percent Percent Percent 1 month at50 o. 85 107 107 107 2 months at 40 C-" V 83 103 106 103 3 months at 50 C 50 100 103 3 months at 40 O 80 102 102 105 3 months at 25 C--- 88. 108 108 p 103 1 year at 40 C 60 95 97 -102 1 year at 25 C 81 '100 .101 105 EXAMPLE 8 (a) A solution containing 1500 units of sodium heparin in 30 cc. of Water containing 0.225% sodium ch1oride and 5% dextrose is stored in a 500 cc. glass container.

(b) The solution of (a) is buttered with 0.2175% sodium citrate and 0.015% citric acid with a resulting pH of 6.2, and stored in a glass container.

(0) The control solution (a) is buttered with 0.326% sodium citrate and 0.0225 citric acid with a resulting pH of 6.2 and stored in a glass container.

The initial activity of the solutions is 96%.

Table VIII 1 Period (41) (b) (c) Percent Percent Percent 2 months at 50 0-- 76 98 6 months at' 25 O 94 6 months at 40 C 84 6 months at 50 C 50 0 months at 25 C 88 9 months at 40 C- v 1 year at 40 C EXAMPLE 9 A solution containing 1000-units of sodium heparin and 1.0 g. of dextrose in 20 cc. of water is observed on storage in 50 cc. glass containers containing sufficient shredded plasticized polyvinylchloride each to produce a surface area comparable to a polyvinylchloride bag of the same content. The following three conditions are checked:

(a) Unbufiered control.

(b) Buffered with 0.25% w./v. of sodium citrate and 0.03% w./v. of citric acid to a pH of 5.5.

Table IX Period (a) Percent Percent Percent 1 month at 40 0... 1 month at 50 0... 2 months at 50 C 3 months at 25 0-- 3 months at 40 O 3 months at 50 C 5 months at 50 C 6 months at 25 0 6 months at 40 0 In a similar test, the above solution is stored under identical original container and solution conditions but buffered with 0.2l75% w./v. of sodium citrate and 0.015% citric acid to a pH of 6.2 at 25 and 40 C. respectively. After 9 months the activities are found to be 94% and 91% respectively.

From the above examples it will be seen that admixing small amounts of citric acid and sodium citrate results in stabilization of the sodium heparin solutions for storage in plastic containers ortogether with other physiologically active ingredients. Particularly striking is the great improvement when compared to the control samples stored in plastic containers at elevated temperatures. Thus, an unbufiered sodium heparin solution deteriorates rapidly at 50 C. or above, whereas the buifered solutions remain substantially unchanged.

For practical reasons, the above examples only demonstrate the use of plasticized polyvinylchloride containers. However, it will be obvious to those skilled in the art that other polymers decomposing with evolution of acidic components will affect the sodium heparin solution in the same fashion. Among those other polymers are poly- (vinylidene chloride), unplasticized polyvinylchloride, and copolymers of these materials as Well as similar plastic materials. Obviously, similar heparin solutions may be stabilized in the identical fashion, i.e., those within concentrations of from about 10 to about 50,000 units per milliliter, etc.

It has also been demonstrated that the buffering action of sodium citrate and citric acid is efiective when other components are mixed with the heparin solution. The most common of these additives is dextrose, but other physiologically active ingredients sometimes included in heparin solutions may cause similar losses of activity of the heparin and their destructive effect can also be counteracted with the inclusion of the bufier of the present invention.

As shown in the examples above, the addition of sodium citrate produces a stable heparin solution whether it is mixed with a minor amount of citric acid or not. Although ordinarily the addition of'citric acid may be desirable, its presence is only indicated to adjust the sodium heparin solution to a particular pH. The most desirable range for injectable solution is from a pH of 8.0 to a pH of 4.5. A preferred pH range for such an injectable solution lies between about 6.0 and 7.5.

Others may practice the invention in any of the numerous ways which will be suggested to one skilled in the art by the present disclosure. All such practice of the invention is considered to be a part hereof provided it falls within the scope of the appended claims.

I claim:

1. A stable, physiological heparin solution suitable for storage in a plastic container containing dissolved therein a stabilizing amount of sodium citrate and having a pH of between 4.5 and 8.0, said pH being adjusted with the required amount of citric acid.

2. A stable, physiological heparin solution suitable for storage in a plastic container containing dissolved therein from 0.01 to 0.5% w./v. of sodium citrate, said solution having a pH of between 4.5 and 8.0, said pH being adjusted with the required amount of citric acid.

3. The solution of claim 2 wherein said pH is adjusted with citric acid to from 6.0 to 7 .5

4. A stable, physiological heparin solution suitable for storage in a plastic container containing dissolved therein from about 10,000 to about $0,000,000 units of heparin, about 9 grams of sodium chloride, from about 0.1 to 5 grams of sodium citrate per liter of vsolution and sufficient citric acid to adjust the pH to from 4.5 to 8.0.

5. The solution of claim 4 wherein said pH is adjusted with citric acid to from 6.0 to 7 .5

6. A stable, physiological heparin solution suitable for storage in a plastic container containing dissolved therein from about 10,000 to about 50,000,000 units of heparin, about 50 grams of dextrose, from about 0.1 to 5 grams of sodium citrate per liter of solution and sufficient citric acid to adjust the pH to from 4.5 to 8.0.

7. The solution of claim 6 wherein said pH is adjusted With citric acid to from 6.0 to 7.5.

8. A stable, physiological heparin solution suitable for storage in a. plastic container containing dissolved therein a stabilizing amount of sodium citrate and having a pH of between 4.5 and 8.0, said pH being adjusted with the required amount of citric acid, said solution being stored in a polyvinylchloride container.

OTHER REFERENCES Kent: Biochemistry of the Aminosugars, 1955 Academic Press, London, England, page 88, 

1. A STABLE PHYSIOLOGICAL HEPARIN SOLUTION SUITABLE FOR STORAGE IN A PLASTIC CONTAINER CONTAINING DISSOLVED THEREIN A STABILIZING AMOUNT OF SODIUM CITRATE AND HAVING A PH OF BETWEEN 4.5 AND 8.0, SAID PH BEING ADJUSTED WITH THE REQUIRED AMOUNT OF CITRIC ACID. 